Durand_185-187.qxd INTRODUCTION Pastures are continually contaminated by the free- living stages of various species of parasitic nema- todes which infect domestic livestock. The resist- ance of one such parasite, Haemonchus contortus, to anthelmintics in South Africa and other parts of the world, led to the formulation of alternative strategies for its control. Among these was the use of nematophagous fungi such as Duddingtonia fla- grans for biological control of infective larvae on pastures. Chlamydospores of D. flagrans, fed to livestock, survive the digestive processes and are viable when voided in the faeces. Parts of the fungal mycelium growing from germinating chlamydospores become modified to form three-dimensional adhesive nets which trap and then feed on nematode larvae. On contaminated pastures, D. flagrans has been shown to be effective in reducing the number of infective larvae of H. contortus and Trichostrongylus colubri- formis from sheep, Ostertagia ostertagi from calves, strongyles from horses, and Oesophagostomum dentatum and Hyostrongylus rubidus from pigs (Larsen 2000). The aim of this study was to find a South African strain of D. flagrans adapted to local environmental 185 Onderstepoort Journal of Veterinary Research, 72:185–187 (2005) RESEARCH COMMUNICATION Survey of nematophagous fungi in South Africa D.T. DURAND1*, H.M. BOSHOFF2, L.M. MICHAEL3 and R.C. KRECEK4 ABSTRACT DURAND, D.T., BOSHOFF, H.M., MICHAEL, L.M. & KRECEK, R.C. 2005. Survey of nematophagous fungi in South Africa. Onderstepoort Journal of Veterinary Research, 72:185–187 Three hundred and eighty-four samples of leaf litter, soil, faeces from domestic and game animals, compost and aqueous cultures of infective nematode larvae contaminated with unidentified fungi were plated out on water agar, baited with pure infective larvae of Haemonchus contortus, incubat- ed and examined for the presence of nematophagous fungi. Duddingtonia flagrans was isolated from five samples, and 73 samples were positive for other nem- atophagous fungi. Keywords: Arthrobotrys oligospora, Duddingtonia flagrans, Haemonchus contortus, nematophagous fungi * Author to whom correspondence is to be directed 1 Department of Veterinary Tropical Diseases, Faculty of Vet- erinary Science, University of Pretoria, Private Bag X04, Onderstepoort, 0110 South Africa 2 Department of Veterinary Tropical Diseases, Faculty of Vet- erinary Science, University of Pretoria, Private Bag X04, Onderstepoort, 0110 South Africa. Present address: P.O. Box 81989, Doornpoort, 0017 South Africa 3 Division of Parasitology, Agricultural Research Council, Onderstepoort Veterinary Institute, Private Bag X05, Onder- stepoort, 0110 South Africa. Present address: Bayer (Pty) Ltd, P.O. Box 143, Isando, 1600 South Africa 4 Department of Veterinary Tropical Diseases, Faculty of Vet- erinary Science, University of Pretoria, Private Bag X04, Onderstepoort, 0110 South Africa. Present address: Depart- ment of Zoology and Entomology, Faculty of Natural and Agricultural Sciences, University of Pretoria, Pretoria, 0002 South Africa Accepted for publication 24 January 2005—Editor conditions that could be used in an integrated worm control programme. In addition (although not the pri- mary aim), the occurrence of other nematophagous fungi in the study area was also recorded. MATERIALS AND METHODS The modified sprinkling technique of Larsen, Wol- strup, Henriksen, Dackman, Grønvold & Nansen (1991) was used for the isolation of nematophagous fungi. Soil, faeces, leaf litter and compost samples collected from five provinces in South Africa (Gau- teng, Mpumalanga, North West, Limpopo and Free State) were sprinkled in a cross configuration on individual plates of 2 % water agar containing 0.02 % tetracycline hydrochloride to suppress bac- terial growth. Approximately 3 ml of aqueous cul- tures of infective nematode larvae contaminated with unidentified fungi were pipetted onto the surface of plates containing the same medium. The plates were incubated at 26 °C, baited at least twice a week for 3 weeks with approximately 3 500 infective H. con- tortus larvae from a pure aqueous culture and exam- ined for signs of nematophagous activity every 2–3 days. The conidia and chlamydospores of some nematophagous isolates were photographed using light microscopy or scanning electron microscopy (SEM). Others were measured and, in some cases, drawn to facilitate identification. Identification was based on the descriptions of type strains (De Hoog 1985; Rubner 1996). The criteria included the type of structure used to trap larvae, the shape, size and numbers of conidia, the configuration of the conid- iophore, and the presence or absence of chlamy- dospores in mature fungal cultures. Conidia grow- ing from trapped larvae on water agar plates were transferred by means of sterile glass inoculation needles to corn meal agar (Difco® Laboratories) plates, to purify the isolate. Pure cultures of six isolates of nematophagous fungi were sent to the “Centraalbureau voor Schim- melcultures” in the Netherlands for expert identifi- cation, to either confirm our findings or identify iso- lates that were problematic. Conidia and chlamydospores of D. flagrans were prepared for SEM as follows: 1 cm square blocks of corn meal agar cultures were vapour-fixed in osmi- um tetroxide for 12 h and air-dried for 2 days. The air-dried blocks were mounted on aluminium stubs, sputter coated with gold and viewed in a Philips XL 20 scanning electron microscope. RESULTS Five isolates of D. flagrans and 73 isolates of other nematophagous fungi were obtained from 384 cul- tures of soil, faeces, compost, leaf litter and aque- ous suspensions of infective larvae contaminated with unidentified fungi. The samples were collected within a 500 km radius of Pretoria, South Africa. The localities included agricultural land, bushveld, urban gardens, nature reserves and a commercial compost processing plant. The most common nematophagous fungus isolated was Arthrobotrys oligospora. The other nemato- phagous fungi isolated were Arthrobotrys superba, Arthrobotrys dactyloides, Arthrobotrys botryospora, Arthrobotrys scaphoides and Monacrosporium gephyropagum. Two isolates of D. flagrans were isolated from compost and three from leaf litter. 186 Nematophagous fungi in South Africa FIG. 1 SEM of conidia from a South African isolate of Dudding- tonia flagrans FIG. 2 SEM of a chlamydospore from a South African isolate of Duddingtonia flagrans DISCUSSION Twenty percent of the samples cultured were posi- tive for nematophagous fungi and D. flagrans has for the first time been isolated in Africa. This pro- vides the opportunity to compare the local fungus with strains from elsewhere in the world as regards their efficacy in reducing infective parasitic larvae on pastures. The doses of D. flagrans chlamydo- spores required to significantly reduce the number of infective larvae contaminating pasture, are in the order of 105–106 chlamydospores per kilogram body mass per day (Peña, Miller, Fontenot, Gilles- pie & Larsen 2002) and the residual effect is limited to 2 days (Waller, Knox & Faedo 2001). Locally iso- lated strains of D. flagrans would need to be cultured in bulk to obtain sufficient quantities of chlamydo- spores for further studies under African conditions. Duddingtonia flagrans grows well on cereals and approximately 250 000 chlamydospores have been counted on the surface of a single grain of barley (Grønvold, Wolstrup, Nansen, Henriksen, Larsen & Bresciani 1993). It has been shown that there is very little genetic variation in isolates of D. flagrans from Denmark, the United Kingdom, France, Germany, the United States of America, Australia, Malaysia and India (Faedo 2001). Faedo (2001) suggests that it is pos- sible that D. flagrans from countries in Europe was introduced to Asia and Australia in exported live- stock. The South African strains of D. flagrans had not yet been isolated at the time of Faedo’s study but the same hypothesis applies. Chlamydospores from a strain of D. flagrans isolated in Denmark have been produced commercially. This survey has demonstrated the presence of local strains of D. fla- grans in South Africa and together with the limited genetic variation between isolates from different parts of the world, should address any concerns about importing a foreign fungus into this country. Recent studies in Australia have been focused on finding a practical method of delivery of D. flagrans chlamydospores to livestock, for use by large scale commercial farmers (Waller, Faedo & Ellis, 2001; Waller, Knox & Faedo 2001). ACKNOWLEDGEMENTS The authors wish to thank the following people and organizations: Dr Michael Larsen and Dr Kobus van der Merwe for their help and encouragement, Ms D. Josling for the SEM preparations and photographs, Just Nature Organics, Onderstepoort and Mr Hans Heilgendorff of the National Botanical Gardens (Pre- toria) for their cooperation, the Food and Agricultural Organization, U.N, Virbac RSA, and the Department of Veterinary Tropical Diseases, Faculty of Veteri- nary Science, University of Pretoria, for financial support. REFERENCES DE HOOG, G.S. (Ed.) 1985. Taxonomy of the Dactylaria com- plex, IV–VI. Studies in Mycology, 26:1–122. FAEDO, M. 2001. Growth, trapping and genetic diversity of Duddingtonia flagrans—a biological control agent of free-liv- ing larval stages of ruminant parasitic nematodes. Ph.D the- sis, Danish Centre for Experimental Parasitology, The Royal Veterinary and Agricultural University, Denmark. 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